d-Mandelate dehydrogenase (DMDH) has the potential to convert d-mandelic acid to phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food additives. A novel NAD-dependent d-mandelate dehydrogenase was cloned from Lactobacillus harbinensi (LhDMDH) by genome mining and expressed in Escherichia coli BL21. After being purified to homogeneity, the oxidation activity of LhDMDH toward d-mandelic acid was approximately 1200 U·mg, which was close to four times the activity of the probe. Meanwhile, the k/ K value of LhDMDH was 28.80 S·mM, which was distinctly higher than the probe. By coculturing two E. coli strains expressing LhDMDH and LcLDH, we developed a system for the efficient synthesis of PGA, achieving a 60% theoretical yield and 99% purity without adding coenzyme or cosubstrate. Our data supports the implementation of a promising strategy for the chiral resolution of racemic mandelic acid and the biosynthesis of PGA.
Phenylglyoxylic
acid (PGA) are key building blocks and widely used to synthesize pharmaceutical
intermediates or food additives. However, the existing synthetic methods
for PGA generally involve toxic cyanide and complex processes. To
explore an alternative method for PGA biosynthesis, we envisaged cascade
biocatalysis for the one-pot synthesis of PGA from racemic mandelic
acid. A novel mandelate racemase named ArMR showing
higher expression level (216.9 U·mL–1 fermentation
liquor) was cloned from Agrobacterium radiobacter and identified, and six recombinant Escherichia coli strains were engineered to coexpress three enzymes of mandelate
racemase, d-mandelate dehydrogenase and l-lactate
dehydrogenase, and transform racemic mandelic acid to PGA. Among them,
the recombinant E. coli TCD 04, engineered
to coexpress three enzymes of ArMR, LhDMDH, and LhLDH, can transform racemic mandelic
acid (100 mM) to PGA with 98% conversion. Taken together, we provide
a green approach for one-pot biosynthesis of PGA from racemic mandelic
acid.
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